Antifreeze proteins (AFPs) have been identified in a number of organisms, such as fish, plants, and insects, to allow them to survive at subfreezing temperatures. They are all characterized by their ability to depress the freezing point of the solution without appreciably altering the melting point thereby producing a thermal hysteresis. Insect AFPs are often the most active AFPs (10-100 times more active than type I fish AFPs). Enhancers have been identified in AFPs from the beetle Dendroides canadensis (DAFPs) to achieve the optimal antifreeze activity of DAFP. Citrate is the best enhancer that has been identified for DAFPs so far. Although many low molecular weight enhancers have been identified and some of them do play a role in enhancing the antifreeze activity of DAFPs physiologically, the molecular mechanism(s) of the enhancer(s) is not well developed. The study of AFPs and enhancers is essential to advance our understanding of the biological system and facilitate their potential biomedical applications, such as cryopreservation and cryosurgery, and many other applications (e.g., in frozen foods). In this project, we will correlate the structures with the physicochemical properties of selected citrate derivatives. We propose that the efficiency of enhancers on DAFP antifreeze activity depends on the physiochemical properties of the enhancers. We will characterize the enhancement efficiency of selected citrate derivatives on DAFP antifreeze activity and investigate how the enhancement efficiency of the systematically varied citrate derivatives varies with their differing physicochemical properties. To reveal the insights of the molecular mechanism of low molecular weight enhancers, we will then explore the possible interactions among the enhancers, DAFP, and ice. A molecular basis for identifying and rationally designing highly efficient enhancers can be thereby developed. This proposed project offers a new approach to the mechanistic study of AFP enhancers. In addition, the delineation of the molecular mechanism of AFP enhancers will resolve conflicts among published mechanisms of AFPs. The proposed study of AFPs and enhancers is essential to advance our understanding of the biological system. The results of the study will contribute to public health in facilitating the potential biomedical applications of AFPs and enhancers such as in longer storage of transplant organs, cryopreservation, and cryosurgery, and many other applications (e.g., in frozen foods).